In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells.

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that are thought to be responsible for therapy resistance, recurrence, and metastasis through their capacity to self-renew and differentiate into heterogeneous downstream lineages of cancer cells. Understanding the features of CSCs is crucial for managing cancer disease and establishing potential targeted therapeutics. Tumor sphere formation assay is a widely used in vitro method that selects and enriches the CSC subpopulation from the total population of cancer cells, based on their inherent ability to grow and clonally expand in serum-free and nonadherent culture conditions. Here we provide a detailed methodology to generate and propagate spheres from isolated cell suspensions of tumor tissues and cell lines using a semisolid MatrigelTM-based three-dimensional (3D) culture system.

Prostate Cancer Organoids for Tumor Modeling and Drug Screening.

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

Tissue-specific cancer stem/progenitor cells: Therapeutic implications.

Surgical resection, chemotherapy, and radiation are the standard therapeutic modalities for treating cancer. These approaches are intended to target the more mature and rapidly dividing cancer cells. However, they spare the relatively quiescent and intrinsically resistant cancer stem cells (CSCs) subpopulation residing within the tumor tissue. Thus, a temporary eradication is achieved and the tumor bulk tends to revert supported by CSCs' resistant features. Based on their unique expression profile, the identification, isolation, and selective targeting of CSCs hold great promise for challenging treatment failure and reducing the risk of cancer recurrence. Yet, targeting CSCs is limited mainly by the irrelevance of the utilized cancer models. A new era of targeted and personalized anti-cancer therapies has been developed with cancer patient-derived organoids (PDOs) as a tool for establishing pre-clinical tumor models. Herein, we discuss the updated and presently available tissue-specific CSC markers in five highly occurring solid tumors. Additionally, we highlight the advantage and relevance of the three-dimensional PDOs culture model as a platform for modeling cancer, evaluating the efficacy of CSC-based therapeutics, and predicting drug response in cancer patients.

MicroRNAs as clinical tools for diagnosis, prognosis, and therapy in prostate cancer.

Prostate cancer (PCa) is one of the most commonly diagnosed cancers among men worldwide. Despite the presence of accumulated clinical strategies for PCa management, limited prognostic/sensitive biomarkers are available to follow up on disease occurrence and progression. MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression through post-transcriptional regulation of their complementary target messenger RNA (mRNA). MiRNAs modulate fundamental biological processes and play crucial roles in the pathology of various diseases, including PCa. Multiple evidence proved an aberrant miRNA expression profile in PCa, which is actively involved in the carcinogenic process. The robust and pleiotropic impact of miRNAs on PCa suggests them as potential candidates to help more understand the molecular landscape of the disease, which is likely to provide tools for early diagnosis and prognosis as well as additional therapeutic strategies to manage prostate tumors. Here, we emphasize the most consistently reported dysregulated miRNAs and highlight the contribution of their altered downstream targets with PCa hallmarks. Also, we report the potential effectiveness of using miRNAs as diagnostic/prognostic biomarkers in PCa and the high-throughput profiling technologies that are being used in their detection. Another key aspect to be discussed in this review is the promising implication of miRNAs molecules as therapeutic tools and targets for fighting PCa.

Novel therapeutic diiminoquinone exhibits anticancer effects on human colorectal cancer cells in two-dimensional and three-dimensional in vitro models.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality. Cancer stem cells (CSCs) in CRC, which are spared by many chemotherapeutics, have tumorigenic capacity and are believed to be the reason behind cancer relapse. So far, there have been no effective drugs to target colon CSCs. Diiminoquinone (DIQ) has shown promising effects on targeting colon cancer. However, there is limited research on the effects of DIQ on eradicating CSCs in CRC. AIM: To investigate the anticancer potential of DIQ on colon CSCs in two-dimensional (2D) and three-dimensional (3D) models using colonospheres and patient-derived organoids. METHODS: Various 2D methods have been used to assess the effect and the mechanism of DIQ on HCT116 and HT29 cell lines including cell proliferation and viability assays, migration and invasion assays, immunofluorescence staining, and flow cytometry. The potency of DIQ was also assessed in 3D culture using the sphere formation assay and colon cancer patient-derived organoid model. RESULTS: Our results showed that DIQ significantly inhibited cell proliferation, migration, and invasion in HCT116 and HT29 cell lines. DIQ treatment induced apoptosis along with an accumulation of HCT116 and HT29 cancer cells in the sub-G1 region and an increase in reactive oxygen species in both CRC cell lines. DIQ reduced sphere-forming and self-renewal ability of colon cancer HCT116 and HT29 stem/progenitor cells at sub-toxic doses of 1 µmol/L. Mechanistically, DIQ targets CSCs by downregulating the main components of stem cell-related -catenin, AKT, and ERK oncogenic signaling pathways. Potently, DIQ displayed a highly significant decrease in both the count and the size of the organoids derived from colon cancer patients as compared to control and 5-fluorouracil conditions. CONCLUSION: This study is the first documentation of the molecular mechanism of the novel anticancer therapeutic DIQ via targeting CSC, a promising compound that needs further investigation.

Drug resistance in metastatic castration-resistant prostate cancer: an update on the status quo.

Prostate cancer (PCa) is a leading cause of cancer-related morbidity and mortality in men globally. Despite improvements in the diagnosis and treatment of PCa, a significant proportion of patients with high-risk localized disease and all patients with advanced disease at diagnosis will experience progression to metastatic castration-resistant prostate cancer (mCRPC). Multiple drugs are now approved as the standard of care treatments for patients with mCRPC that have been shown to prolong survival. Although the majority of patients will respond initially, primary and secondary resistance to these therapies make mCRPC an incurable disease. Several molecular mechanisms underlie the development of mCRPC, with the androgen receptor (AR) axis being the main driver as well as the key drug target. Understanding resistance mechanisms is crucial for discovering novel therapeutic strategies to delay or reverse the progression of the disease. In this review, we address the diverse mechanisms of drug resistance in mCRPC. In addition, we shed light on emerging targeted therapies currently being tested in clinical trials with promising potential to overcome mCRPC-drug resistance.